mouse antibody mab1696  (GenScript corporation)

Journal: Journal of molecular biology

Article Title: Mechanism of Dissociative Inhibition of HIV Protease and its Autoprocessing from a Precursor

doi: 10.1016/j.jmb.2012.05.024

Figure Lengend Snippet: Inhibition of autoprocessing of TFR-PR-RT20 by scFv. (A) Time course (in hours) of the reaction (5.17 µM precursor) at pH 6.5 in 0.5x PBS and 0.2 M urea. M denotes molecular weight standards in kDa. (B) Autoprocessing in the absence and presence of ~4-fold molar excess of scFv. Reactions and gel analysis were carried out as described in Experimental procedures. Proteins were visualized by Coomassie staining. (C) Inhibition of autoprocessing using mAb1696 monitored by western blotting. Lanes 1 and 2 are controls without scFv; lanes 3 and 4, with same excess of scFv as in B. Note that the mAb does not recognize the precursor containing the TFR (time zero lanes) but recognizes the precursor only after the cleavage at its N-terminus. The scheme accounts for the experimental results shown in panels B and C. The stepwise maturation of PR from a model precursor, comprising the PR (red) flanked by the transframe region (TFR, green) and a partial reverse transcriptase sequence (ΔRT, black), and its inhibition by scFv are shown.

Article Snippet: Proteins were separated on 10–20% Tris-tricine gels and immunoblotted either using the mouse antibody mAb1696 according to the One-Hour Western Detection System provided by Genscript or as described previously 36 using the human antibody PRM1.

Techniques: Inhibition, Molecular Weight, Staining, Western Blot, Reverse Transcription, Sequencing

Journal: Journal of molecular biology

Article Title: Mechanism of Dissociative Inhibition of HIV Protease and its Autoprocessing from a Precursor

doi: 10.1016/j.jmb.2012.05.024

Figure Lengend Snippet: Western blotting of mAb1696 binding to mature PR and its precursor mimetics. Lane identifications and notation are described in Table 1. Proteins (500 ng/lane) were subjected to electrophoresis on 10–20% gradient Tris-Tricine gels, transferred to nitrocellulose membrane and probed with mAb1696 as described in the Experimental procedures. Only those proteins lacking an N-terminal flanking sequence are recognized by mAb1696. Proteins ranging in concentration from 0.28–0.6 mg/mL are shown in panels B and E by Coomassie staining to validate the western blotting results (a and c). Arrows in panel A correspond to stained band positions seen in panel B.

Article Snippet: Proteins were separated on 10–20% Tris-tricine gels and immunoblotted either using the mouse antibody mAb1696 according to the One-Hour Western Detection System provided by Genscript or as described previously 36 using the human antibody PRM1.

Techniques: Western Blot, Binding Assay, Electrophoresis, Membrane, Sequencing, Concentration Assay, Staining